Construction and Expression of a Bifunctional Single-Chain Antibody againstBacillus cereusSpores

Abstract

The variable-region genes of monoclonal antibody againstBacillus cereus spores were cloned from mouse hybridoma cells by reverse transcription-PCR. The heavy- and light-chain variable-region genes were connected by a 45-base linker DNA to allow folding of the fusion protein into a functional tertiary structure. For detection of protein expression, a 10-amino-acid strep tag (biotin-like peptide) was attached to the C terminus of recombinant antibody as the reporter peptide. The single-chain antibody construct was inserted into the expression vector and expressed in Escherichia coliunder the control of the T7 RNA polymerase-T7 promoter expression system. The expressed single-chain antibody was detected on Western blots by using a streptavidin-conjugated enzyme system. This small recombinant antibody fragment (ca. 28,000 Da by calculation) hadB. cereus spore binding ability and antigen specificity similar to those of its parent native monoclonal antibody.

Development of a Streptavidin-Conjugated Single-Chain Antibody That Binds Bacillus cereus Spores

“…4). The unexpected cross-reaction with B. subtilis A spores observed with the strep tag (a 10-amino-acid peptide)-conjugated single-chain antibody that has the same primary structure for the antigen-binding domains (19) was not detected in this single-chain antibody. A simple competitive dot blotting immunoassay demonstrated that the recombinant and parent monoclonal antibodies bind to the same or very similar epitopes.…”

“…The plasmid pGEM-3Z, which was used for general cloning and sequencing purposes, was obtained from Promega. The pET22b(ϩ)-derived plasmid pET22IgTag (19) was used as the single-chain antibody gene source. This plasmid contains a T7/lac promoter (37), a pelB signal sequence (16) for protein relocation, the complete anti-B.…”

“…The overnight culture was transferred into plasmid medium (24) containing ampicillin (500 g/ml) and incubated at 37°C for 5 h with shaking, and then protein expression was induced by the addition of 0.2 mM isopropyl-␤-D-thiogalactopyranoside (IPTG; GIBCO BRL) and by incubation for 2 h at 37°C. The cellular location of the fusion protein was analyzed using the procedures as previously described (19). The periplasmic and cytoplasmic proteins were extracted by osmotic shock and cell lysis (lysozyme treatment and sonication) procedures, respectively.…”

“…It was connected with the gene of single-chain anti-B. cereus spore antibody (19) to form a fusion protein gene. This bifunctional single-chain antibody gene was expressed by Escherichia coli.…”

A Simple Methodology for Conversion of Mouse Monoclonal Antibody to Human-Mouse Chimeric Form

“…Mouse Ig-Primer set is also commercially available. For example, the one from Novagen has been successfully applied in many previous studies (e.g., [18][19][20][21]).…”